HTLV-I and HTLV-II are oncogenic retroviruses associated with neoplastic transformation of CD4+ and CD8+ T-lymphocytes. These viruses display distinct pathogenic properties in vivo; however, both viruses infect and transform primary human T-cells in cell culture. The viral trans-activator protein, Tax, has been implicated in the HTLV oncogenic process, primarily due to its ability to aberrantly activate T-cell genes involved in growth regulation. However, conclusive evidence for the role of Tax in the induction of malignancy is lacking because of the inability to mutate tax in the context of an infectious virus in order to dissociate viral replication from cellular transformation. To circumvent this problem, a mutant HTLV-II (HTLV C-enh), which replicates by a Tax independent mechanism, was constructed. This unique chimeric virus maintains the capacity to efficiently transform primary T-cells; however, a replication-competent tax-knockout of this virus fails to transform T-cells indicating that Tax is required for T-cell transformation by HTLV. Domains of Tax important for HTLV-mediated transformation of human T-cells have not been identified nor has the physiological role of phosphorylation in Tax function been ascertained. The novel HTLV- c-enh provides a unique opportunity to study Tax flinction in an infectious virus system. Three integrated specific aims are proposed to precisely determine the molecular basis of T-cell transformation, T-cell tropism, and induction of leukemia by HTLV. In Specific Aim 1, a functional analysis of HTLV-II-tax will identify mutants that fail to transactivate CREB/ATF or NFkappaB/Rel signaling pathways, hypothesized to be important for transformation, and determine the functional significance of Tax phosphorylation. In Specific Aim 2, HTLV C-enh mutant viruses will be used to determine the precise mechanism by which Tax mediates transformation of human T- cells. An HTLV-I that replicates by a Tax-independent mechanism will be generated for comparative studies. In Specific Aim 3, the molecular basis for pathogenic differences between HTLV-I and HTLV-II, with specific emphasis on CD4+ and CD8+ T-cell transformation tropism, will be determined using HTLV-I/HTLV-II recombinants and transformation assays. The proposed experiments will provide fundamental information about functional determinants of HTLV-II Tax, on HTLV-I and HTLV-II biology, and the mechanism of HTLV transformation and pathogenesis. Moreover, these studies will contribute to a better understanding of T-cell activation and differentiation. This information may be critical for future therapeutic approaches for cancers associated with these viruses as well as other types of T-cell leukemias and lymphomas.